Conversion of Industrial Sweetpotatoes for the Production of Ethanol

ABSTRACT

DUVERNAY, WILLIAM HAUSER. Conversion of Industrial Sweetpotatoes for the

Production of Ethanol. (Under the direction of Mari S. Chinn.)

Starch is a renewable complex carbohydrate currently being used to produce ethanol from

corn. Although corn starch to ethanol is a mature process, corn production is not feasible for

every region of the United States. Sweetpotatoes (

Ipomoea

batatas

, Morning-glory family)

are a low-impact crop grown primarily in the southeast region of the U.S. and offer a viable,

alternative starchy raw material that can be converted to useful sugar feedstocks needed for

production of ethanol and other value added products. The process of converting

sweetpotato starch into ethanol can be described in three basic steps: liquefaction using

α-amylase or some other liquefying agent to gelatinize available starch, saccharification using

saccharifying enzymes to convert gelatinized starch into soluble sugars, and fermentation of

the sugars into ethanol. The overall goal of this project was to generate the information

necessary to define an environmentally friendly process for conversion of industrial

sweetpotatoes (ISPs) into simple sugars and ethanol. Specific objectives included: 1)

Examining liquefaction, saccharification, and fermentation of FTA-94 ISPs using α-amylase

and glucoamylase for the production of ethanol; and 2) Examining the enzymatic hydrolysis

and fermentation of ISPs with the addition of pullulanase for ethanol production. The

significance of enzyme loading rate, incubation time, and temperature on changes in starch

content and sugar concentrations during each hydrolysis step was evaluated. Ethanol

α-Amylase (Liquozyme SC) at different loading rates (0.045, 0.45, and 4.5% KNU-S/g

dry ISP ) was tested during liquefaction over time at 85°C; three glucoamylases (Spirizyme

Fuel, Spirizyme Plus Tech, and Spirizyme Ultra) were tested during saccharification at 65˚C

at different loading rates (0.5, 1.0, and 5.0 AGU/g dry ISP) over time. Results showed that

the majority of available starch, 47.7 and 65.4% of dry matter, was converted during

liquefaction of flour and fresh sweetpotato preparations, respectively, with the addition of

0.45 KNU-S/g dry ISP of Liquozyme SC after 2 hours incubation (66.4 and 80.1% initial

starch contents, respectively). Saccharification was able to increase the breakdown of starch,

but its primary function was conversion of short chain carbohydrate polymers to fermentable

sugars. The addition of 5.0 AGU/g of Spirizyme Ultra after 48 hours was able to produce

795.4 and 685.3 mg of glucose/g of starch with flour and fresh preparations, respectively.

Yeast fermentation on hydrolyzed starch was examined over time with and without the

addition of salt nutrients. Yeast were able to produce 62.6 and 33.6 g/L of ethanol for flour

(25% w/v substrate loading) and fresh (12.5% w/v) ISP, respectively, after 48 hours without

salts.

Pullulanase (Promozyme) was added to the saccharification step to enhance hydrolysis of

α-1,6 bonds in ISP starch polymers and increase glucose yields. The addition of Promozyme

after 48 hours of saccharification with 5.0 AGU/g Spirizyme Ultra at 45, 55, and 65ºC

showed no consistent increase in the change in starch content. Adding 0.5 NPUN/g of

Promozyme to 5.0 AGU/g Spirizyme during saccharification at 55ºC slightly increased the

Fermentation of sugars produced from this treatment combination yielded 310.7 and 333.3

mg ethanol/g dry ISP for flour and fresh preparations.

Results of these experiments show that sweetpotatoes can be converted to glucose

through enzymatic methods, hydrolyzing up to 88% of available starch, and that produced

sugars are fermentable by yeast. Based on the data and ISP production yields in North

Carolina, it is possible to generate at least 700 gallon of ethanol per acre of ISPs. However,

challenges still need to be addressed related to improved conversion efficiency, scale up, and

Conversion of Industrial Sweetpotatoes for the Production of Ethanol

by

William Hauser Duvernay

A thesis submitted to the Graduate Faculty of

North Carolina State University

in partial fulfillment of the

requirements for the Degree of

Master of Science

Biological and Agricultural Engineering

Raleigh, North Carolina

2008

APPROVED BY:

_____________________________ ______________________________

G. Craig Yencho Ratna R. Sharma-Shivappa

_____________________________

Mari S. Chinn

ii

BIOGRAPHY

William “Billy” Hauser Duvernay was born on March 23, 1984, in the great city of New

Orleans, Louisiana. He is the second son of Thomas and Sarah Duvernay, and the younger

brother of Matthew Duvernay. Billy spent the first half of his life in New Orleans, playing

baseball, basketball, and football at Lakeview playground just about every free second that

wasn‟t spent in school. The remainder of his time was split up between fishing, family

vacations, and basically doing everything his older brother told him to do.

Just before Billy entered high school, the Duvernay family made the 26 mile move north

above Lake Pontchartrain to the wooded suburbs of the city (less football and basketball;

more baseball and fishing). Billy graduated from St. Paul‟s High School second in his class

in 2002, where he went on to study Biological Engineering at Louisiana State University.

His competitive edge was kept satisfied on the Ultimate Frisbee Team at LSU. Performing

undergraduate research under the guidance of Dr. Cristina Sabliov, Billy realized the

importance of renewable resources, and decided that he wanted to spend more time

researching solutions to the world‟s energy problems. That‟s when he met Dr. Mari S. Chinn

at North Carolina State University, and after graduating from LSU in 2006, he left his family

and friends to pursue a Master‟s Degree in Raleigh, North Carolina, trying to find alternate

methods for producing ethanol. He‟s been eating, sleeping, and breathing sweetpotatoes

ever since.

After graduate school, Billy plans on moving back to Louisiana where he can be closer to

his family and friends. He intends to pursue a career in the ethanol or bioprocessing industry.

iii

ACKNOWLEDGEMENTS

First and foremost, I‟d like to thank Dr. Mari Chinn for all of her efforts as my chief

committee member. Dr. Chinn, over the past two years, you have helped me to realize my

potential as a scientist/engineer acting as both my mentor and my friend. As my mentor you

challenged me to cover every possible angle and research every possibility. Even when you

knew the answer to your question, which I imagine was more often than not, you forced me

to think my way through the answer. As my friend, you were there to help me whenever I

needed it, whether it was running HPLC until 1 a.m. on a Sunday night for research or just

having a conversation in your office to pass the time. Thank you for everything. I couldn‟t

have done it without you.

I would also like to thank all of my acting committee members: Dr. Sharma, Dr. Yencho,

and Dr. Sosinski. I know I wasn‟t the easiest graduate student, with all of my changing dates

and annoying emails, but your patience and encouragement throughout my stay at N.C. State

will not be forgotten.

Dad, thanks for all of your support over the last two years and for all of my life. Your

Monday morning conversations about “what‟s goin‟ on”, though sometimes a little earlier

than I would have hoped, have kept me in line and focused and provided me with the

motivation to finish my work not only to impress you but to enable me to put our name on

something that I am proud to call my work. And I‟m sure you never tired of answering my

distressed calls about my countless problems, including, “what do I do when a tree falls on

iv

Mom, there‟s nothing that I can really say to express my thanks to you for all of your

help, but I‟ll try. Your numerous letters and care packages over the last two years have kept

me closer to home than I ever thought I could be living over 850 miles from your doorstep.

And I don‟t know what I would have done without our weekly conversations keeping me up

to date on the family news. You have been nothing but understanding and encouraging, and I

can‟t thank you enough.

Matt, all my life I have tried to live up to what I thought your expectations of me might

be. As my older brother, you‟ve always provided me with something to live up to and given

me the encouragement (and sometimes the tutoring) to not only perform, but to excel at

everything I do. As my best friend we‟ve been through the best and worst times together, but

always come out on top and stronger because of it. And now I can finally say I finished

something before you: grad school.

Lisa, thanks for all of your encouragement and support. You have provided me with the

love and encouragement to work every day so that I could one day come home and start my

life with you. Without you there to complain to about what was going wrong and brag to

about what was going right, I wouldn‟t have had the motivation to continue. Thank you for

always being there.

Finally, I‟d like to thank the guys: Hayes, Evan, Arthur, Rob, and Oneal. Without our

camping trips, breaks at the office, and late nights at alibi, those long nights in the lab might

have driven me crazy. Your friendships have meant a lot to me, so let‟s keep the good times

v

TABLE OF CONTENTS

List of Figures

……..………

viii

List of Tables

………

ix

Chapter 1: Background and Literature Review

………...

1

1.1 Nonrenewable and renewable resources

……….

2

1.2 Trends in ethanol consumption and use

………...

3

1.3 Lignocellulosic Materials/Feedstock

………...

7

1.3.1 Sugar platform

………....

8

1.3.2 Thermochemical Platform

………...

11

1.4 Starch-based Feedstocks

………...

12

1.4.1 Starch Crops

………...

12

1.4.2 Starch Composition

………...

13

1.4.3 Starch Conversion

………...

14

1.5 Acid hydrolysis

………..…....

15

1.6 High temperature and pressure

………..

15

1.7 Enzyme hydrolysis

………...

16

1.8 Ethanol Fermentation

…………...……….

19

1.9 Objectives

………....

20

1.10 References

……….

21

Chapter 2: Liquefaction, Saccharification, and Fermentation of

FTA-94 Industrial Sweetpotatoes using α-Amylase and Glucoamylase for

the Production of Ethanol

………...

27

2.1 Introduction

………...

27

2.2 Materials and Methods

………..

31

2.2.1 Enzymes

………

31

2.2.1.1 α-Amylase

………...

31

2.2.1.2 Glucoamylase

………..

31

2.2.2 Industrial Sweetpotato Preparation

………..

31

2.2.3 Experimental Design and Statistical Analysis

………..

32

2.2.4 Liquefaction

……….

33

2.2.5 Saccharification

………...

34

2.2.6 Starch Quantification

………..

34

2.2.7 Fermentation

………

35

2.2.8 Sugar Analysis

………..

36

2.2.8.1 Reducing Sugars

………..

36

vi

2.3 Results

………..

37

2.3.1 Liquefaction

……….

37

2.3.1.1 Starch Quantification

………

37

2.3.1.2 Reducing Sugars

………

40

2.3.2 Saccharification

………...

43

2.3.2.1 Starch Quantification

………

43

2.3.2.2 Soluble Sugars

………...

50

2.3.3 Fermentation

………

59

2.4 Discussion

………...

61

2.4.1 Enzymatic hydrolysis of starch

………...

61

2.4.2 Fermentation to ethanol

………..

69

2.5 References

………...

72

Chapter 3: Enzymatic Hydrolysis of Industrial Sweetpotatoes using

α-Amylase, Glucoamylase, and Pullulanase and Fermentation of Sugars

to Ethanol

………...………...

76

3.1 Introduction

………...

76

3.2 Materials and Methods

………..

80

3.2.1 Enzymes

………

80

3.2.1.1 α-Amylase

………...

80

3.2.1.2 Glucoamylase

………..

80

3.2.1.3 Pullulanase

……….

80

3.2.2 Industrial Sweetpotato Preparation

………..

81

3.2.3 Experimental Design and Statistical Analysis

………..

81

3.2.4 Saccharification

………...

82

3.2.5 Starch Quantification

………..

83

3.2.6 Fermentation

………

83

3.2.7 Saccharification Sugars and Ethanol Concentrations

……….

85

3.3 Results

………..

85

3.3.1 Saccharification

………...

85

3.3.1.1 Starch Quantification

………..

85

3.3.1.2 Glucose Production

………

89

3.3.2 Fermentation

………

91

3.4 Discussion

………....

95

3.4.1 Enzymatic hydrolysis

………..

95

3.4.2 Fermentation to ethanol

………..

100

3.5 References

………...

103

Chapter 4: Saccharification and Fermentation Process Assessment

...

106

vii

Appendix A: SAS

Analyses for Liquefaction of FTA-94 ISP

…………..

111

Appendix A.1 Flour ISP

………...

111

Appendix A.2 Fresh ISP

………...

116

Appendix A.3 Comparison of Flour and Fresh ISP

………..

121

Appendix B: SAS

Analyses for Saccharification of FTA-94 ISP with

Spirizyme Fuel, Spirizyme Plus Tech, and Spirizyme Ultra

……….

132

Appendix B.1 Flour ISP

………...

132

Appendix B.2 Fresh ISP

………...

167

Appendix B.3 Comparison of Flour and Fresh ISP

………..

197

Appendix C: SAS

Analyses for Saccharification of FTA-94 ISP with

Promozyme

………...

240

Appendix C.1 Flour ISP

………...

240

Appendix C.2 Fresh ISP

………...

244

Appendix C.3 Comparison of Flour and Fresh ISP

………..

249

Appendix D: Preliminary Saccharification of FTA-94 ISP

using

Spirizyme Plus Tech

………..

257

viii

LIST OF FIGURES

Figure 2.1

Average change in starch (% of dry matter lost) over time during

liquefaction of flour and fresh FTA-94 industrial sweetpotatoes using Liquozyme

SC: A) flour preparations B) fresh preparations……….…...

41

Figure 2.2

The average change in reducing sugars over time during liquefaction of flour

and fresh FTA-94 industrial sweetpotatoes using Liquozyme SC A) flour

preparations B) fresh preparations……….……....

44

Figure 2.3

Average change in starch (% loss of dry matter) over time during

saccharification of flour FTA-94 industrial sweetpotatoes using three different

glucoamylase enzymes: A) Spirizyme Fuel. B) Spirizyme Plus Tech. C) Spirizyme

Ultra………...

47

Figure 2.4

Average change in starch (% of dry matter lost) over time during

saccharification of fresh FTA-94 industrial sweetpotatoes using three different

glucoamylase enzymes: A) Spirizyme Fuel. B) Spirizyme Plus Tech. C) Spirizyme

Ultra………...

49

Figure 2.5

Concentration of HPLC sugars during

saccharification

of flour FTA-94

industrial sweetpotatoes over time using three different enzymes: A) Spirizyme

Fuel, B) Spirizyme Plus Tech, C) Spirizyme Ultra………...

54

Figure 2.6

Concentration of HPLC sugars during

saccharification

of fresh FTA-94

industrial sweetpotatoes over time using three different enzymes: A) Spirizyme Fuel, B)

Spirizyme Plus Tech, C) Spirizyme Ultra………... 57

Figure 2.7

Ethanol, glucose, and fructose concentrations measured over time during

fermentation of flour and fresh FTA-94 industrial sweetpotatoes: A) Flour, 0.1%

yeast, no salt. B) Flour, 0.1% yeast, salt extract. C) Fresh, 0.1% yeast, no salt. D)

Fresh, 0.1% yeast, salt extract...

62

Figure 3.1

Average change in starch (% loss of dry matter) over time during

saccharification of flour and fresh FTA-94 industrial sweetpotatoes using

Promozyme: A) flour preparations. B) fresh preparation………...

87

Figure 3.2

Concentration of glucose found during

saccharification

of flour and fresh

FTA-94 industrial sweetpotatoes over time using Promozyme: A) flour preparation.

B) fresh preparation………..……….

92

Figure 3.3

Ethanol, glucose, and fructose concentrations measured over time during

fermentation of flour and fresh FTA-94 industrial sweetpotatoes: A) Flour, 0.1%

ix

LIST OF TABLES

Table

2.1

ANOVA table of flour FTA-94 liquefaction for incubation time and enzyme

loading on change in starch content and reducing sugars………...

37

Table 2.2

ANOVA table of fresh FTA-94 industrial sweetpotato liquefaction for

incubation time and enzyme loading on change in starch content and reducing sugars.

39

Table 2.3

ANOVA table of flour and fresh FTA-94 industrial sweetpotato during

saccharification for incubation time, type of glucoamylase, and enzyme loading on

change in starch (% of dry matter lost)……….………..

46

Table 2.4

ANOVA table of flour FTA-94 industrial sweetpotato during saccharification

for incubation time, type of glucoamylase, and enzyme loading on levels of glucose...

53

Table 2.5

ANOVA table of flour FTA-94 industrial sweetpotato during saccharification

for incubation time, type of glucoamylase, and enzyme loading on levels of maltose...

53

Table 2.6

ANOVA table of flour FTA-94 industrial sweetpotato during saccharification

for incubation time, type of glucoamylase, and enzyme loading on levels of

maltotriose…...

53

Table 2.7

ANOVA table of fresh FTA-94 industrial sweetpotato during saccharification

for incubation time, type of glucoamylase, and enzyme loading on levels of glucose...

58

Table 2.8

ANOVA table of fresh FTA-94 industrial sweetpotato during saccharification

for incubation time, type of glucoamylase, and enzyme loading on levels of maltose...

58

Table 2.9

ANOVA table of fresh FTA-94 industrial sweetpotato during saccharification

for incubation time, type of glucoamylase, and enzyme loading on levels of

maltotriose………...

58

Table 3.1

ANOVA table of flour and fresh FTA-94 industrial sweetpotato during

saccharification for incubation temperature and enzyme loading on change in starch...

86

Table 3.2

ANOVA table of flour and fresh FTA-94 industrial sweetpotato during

saccharification for incubation temperature and enzyme loading on glucose...……….

90

Table 3.3

ANOVA table of flour and fresh FTA-94 industrial sweetpotato during

1

Chapter 1: Background and Literature Review

The world‟s consumption of energy has steadily risen every year and continues to

increase on an international level. By the year 2030, the Federal Energy Management

Program expects consumption to grow 71% over that of June 2006 (EERE, 2006). Eight

countries have 81% of the world‟s oil reserves, six countries have 70% of all natural gas

reserves, and 89% of the world‟s coal reserves lay in eight countries (Sayigh, 1999).

On a national level, the United States consumed 19.7 million barrels of petroleum per day

in 2002, totaling one-quarter of world‟s entire oil production. More than half of this oil,

62%, was imported (EERE, 2006). The Energy Information Administration (EIA) projected

total petroleum consumption in 2025 to reach 28.3 million barrels per day, having to depend

on 70% from foreign imports (EIA, 2004). This increasing dependency on foreign oil as

domestic supplies lessen threatens the economic and energy security of America.

To compete with this ever growing trend, an economy based on carbohydrates has been

introduced. It focuses on the production of biobased products derived from renewable

biomass. Bioproducts are products created from plant based resources such as agricultural

crops and forestry residues, dedicated energy crops, trees and grasses. Many of the same

products made from petroleum can be made from these biomass resources. The basic

molecules of petroleum being used for production are hydrocarbons. The basic molecules of

plants, however, are carbohydrates, proteins, and oils. Both can be used to produce a wide

2

1.1 Nonrenewable and renewable resources

Nonrenewable resources are resources which cannot be remade.

Fossil fuels like coal,

crude oil, and natural gas are considered to be nonrenewable resources since they do not

naturally reform at a rate fast enough to be sustainable in our society. In 2002, 86% of all

energy consumed in the United States was from fossil fuels (EERE, 2006). These

nonrenewable resources are being utilized as the world‟s main source of energy and are

therefore depleting at an alarming rate.

In addition to providing the world‟s energy, nonrenewable resources are also used in

some way to manufacture many products on the market today. Some common products

derived from fossil fuels include detergents, synthetic fibers (nylon, polyester, acrylic),

plastics, paints, garden hoses, food additives, cleaning products, pesticides, nail polish,

lipstick, and shampoo (EERE, 2006).

Another cause for concern associated with nonrenewable resource-based fuels is their

high carbon content. When these fuels are burned they release carbon back into the

atmosphere as carbon dioxide. Because the demand for energy has risen so rapidly in the

past and continues to increase, the burning of these fuels is thus resulting in a rise in the

concentration of carbon dioxide in the atmosphere, a major cause of the greenhouse effect.

The increasing demand of energy and depleting nonrenewable resources has led to a shift

of focus to use renewable resources as a main source of energy and product generation.

Power derived from hydro, geothermal, wind, and solar resources as well as biomass-derived

energy could provide national energy security, economic growth, and environmental benefits

3

As of 2003, production of biobased textile fibers, polymers, adhesives, lubricants,

soy-based inks, and other products was estimated at 12.4 billion pounds per year (6.2 million

tons) (Pastor et al., 2003). However, the total production of all biobased and non-biobased

products is in the hundreds of billions of pounds (more than 50 million tons). This provides

incredible opportunity for growth of biobased products both on a national and international

level (EERE, 2006).

In 2003 biomass was the leading source of renewable energy in the United States, mostly

coming from industrial heat and steam production by the pulp and paper industry and

electrical generation with forest industry residues and municipal solid waste (MSW). It

provided 2.9 quadrillion Btu (13.61 kJ) of energy and was the source for 47% of all

renewable energy or 3% of the total energy produced in the United States (EERE, 2006).

1.2 Trends in ethanol consumption and use

The US Energy Policy Act of 2005 (FERC, 2005) stated that by the year 2012, the oil

industry will be required to blend 7.5 billion gallons of renewable fuels into gasoline. A new

mandate issued in December of 2007 stated that this Renewable Fuel Standard (RFS) has

increased to require the production of 36 billion gallons of renewable fuels per year by 2020

(EERE, 2008). The most common renewable fuel is ethanol. Currently, gasoline is blended

with oxygenates. The only two oxygenates currently available are ethanol and methyl

tertiary butyl ether (MTBE). MTBE was used to replace lead in gasoline as an oxygen

enhancer in 1979. Now ethanol is replacing MTBE as a fuel additive because MTBE has

4

When ethanol is blended with gasoline, fuel combustion has been known to improve

tailpipe emissions of CO and unburned hydrocarbons that form smog have been reduced

(Wyman, 1996). Also, by using bioethanol, a net reduction of 60-90% in the levels of carbon

dioxide compared to gasoline-consuming vehicles is possible (Brown et al., 1998). The

performance of an ethanol fueled vehicle is also superior to that of gasoline. Ethanol enables

combustion engines to run at higher compression ratios because of its higher octane rating,

causing an increase in net performance (Wyman, 1996). In addition, the vapor pressure and

heat of vaporization of ethanol are both greater than that of a gasoline engine, resulting in

higher power outputs (Zaldivar et al., 2001). However, because of its oxygen content, pure

ethanol has 33% less energy than gasoline (Kosaric, 1996). Ethanol can be blended with

gasoline at levels of 10%, 20%, and 85%, or used as 100% pure. All gasoline vehicles can

use E10 (10% ethanol). E85, however, must be burned in Flex-fueled vehicles, which have

an oxygen sensor in the fuel line and can adjust the engine timing and fuel injection

(Alternative Fuel Education Review).

In 2005, Brazil produced 4.2 billion gallons of ethanol, increasing from 4.0 billion

gallons in 2004. This ethanol is being produced from the feedstock sugar cane. It is used as

either 22% ethanol blends with gasoline or as neat ethanol fuel containing 100% ethanol

(Mielenz, 2001). Brazil has found amazing success using this feedstock, which is grown all

over the country. Sugarcane is an excellent feedstock; however it can only be grown in

select areas of the Unites States due to its limited climate adaptation and can therefore not be

5

The United States produced 3.9 billion gallons of ethanol in 2005. This increased from

3.4 billion gallons in 2004 (USDA, 2006). There were 101 ethanol plants operating in 21

states as of June 2006 with a total production capacity of 4.8 billion gallons of ethanol per

year, and there are 33 ethanol plants currently under construction. The United States has the

capacity to build enough ethanol production facilities to support its citizens. However,

problems surface when the availability of feedstocks and economics for ethanol production

are analyzed.

The primary limiting factors affecting the commercial production of ethanol are the

availability of biomass and the cost of ethanol. An analysis done by Oak Ridge National

Laboratory indicated that the maximum amount of agricultural residues that could be

collected today is roughly 144 million dry tons per year (EERE, 2006). This translates to a

supply of only 10% of our light duty transportation needs. Because these numbers do not

meet our national demands, new resources for the production of ethanol must be discovered

and implemented.

Recently, the price of ethanol in the United States has followed the price of gasoline plus

the 51 cents per gallon Federal excise tax credit. Spot prices of ethanol, however, have

increased to over 4 dollars per gallon as the demand has increased. But ethanol prices are

eventually expected to decrease as the production increases. Also, as ethanol production

increases to fully replace MTBE and meet the requirements set by the government, the price

of ethanol should reflect its value as a gasoline extender and fluctuate up and down with the

6

In the United States, the primary feedstock being used in ethanol production, accounting

for approximately 97%, is corn (EERE, 2006). Although this starch-based resource is an

excellent source of feedstock to produce ethanol, there is not enough corn produced in the

United States to provide sufficient ethanol fuel to meet the energy demand. Another problem

associated with using corn as the only feedstock to produce ethanol is that the main corn

producing states, and hence most of the ethanol plants, are located in the Midwestern states.

This creates a transportation cost for the corn deficient states, because it cannot grow

everywhere in the country. In addition, there are environmental, social and food and feed

market issues surrounding the sustainability of corn as a feedstock for US ethanol production

needs. Negative environmental impacts arise when fertilizer application and acreage usage

is considered. Corn is an inherently inefficient user of nitrogen, where 40-60% of the

nitrogen is not absorbed by the crop, and N levels in downstream aquatic ecosystems from

corn-dominated landscapes are typically 20 to 40 kg N ha

yr

(Balkcom et al., 2003;

Randall et al., 2003). Therefore, increases in the acreage of corn and the fertilizer application

rates due to rising corn prices leads to increased N and P losses to rivers, lakes streams, and

coastal waters, particularly those located downstream of expanding production areas

(Simpson et al., 2008). Corn being used as a feedstock for ethanol production also competes

with the food industry. Because of the demand for corn, a recent increase from 2 to 5 dollars

a bushel has occurred (Cohen, 2008). Relying completely on the production of ethanol from

corn starch is insufficient due to the competition which arises between the corn cultivation

and the limited agricultural land for both food and feed production (Sun and Cheng, 2002).

7

renewable fuels come from four different categories: conventional biofuel (corn starch),

advanced biofuel (renewable fuel other than corn starch), biomass-based diesel, and

cellulosic biofuel (derived from any cellulose or lignin from renewable biomass) (EERE,

2008).

Additional feedstocks suitable for conversion to renewable fuels need to be identified

for other regions of the United States in order to increase advanced biofuel production

.

1.3 Lignocellulosic Materials/Feedstock

Lignocellulosic biomass as a potential feedstock for the production of ethanol can be

obtained from two potential sources: waste material from processes other than fuel

production such as agriculture and forest products industries, or from energy crops which are

grown specifically for the purpose of fuel production (Lynd, 1996). Most lignocellulosic

biomass is composed of the same three major components: cellulose (30-50%), hemicellulose

(20-30%), and lignin (20-30%). Lignocellulosic biomass is a valuable resource for a future

bioindustry. However, problems emerge when cost effective recovery of these components

is attempted (Pastor et al., 2003).

Cellulose is a chain of six-carbon sugars called glucose joined by β-1,4-glycosidic

linkages. Hydrogen bonds between the chains lead to the formation of sheets lying on top of

one another in a staggered position. This makes cellulose very chemically stable, insoluble,

and difficult to break down (EERE, 2006).

Hemicellulose is a highly branched heteropolymer containing sugar residues such as

hexoses (D-galactose, L-galactose, D-mannose), pentoses (D-xylose, L-arabinose), and

uronic acids (Zalvidar et al., 2001). It strengthens the plant wall by forming a short-chain

8

Lignin is the most abundant aromatic polymer in nature, and is what makes up the woody

part of the lignocellulosic biomass. It is composed primarily of carbon ring structures

interconnected by polysaccharides, which are very valuable chemical intermediates.

Separation and recovery of these lignin structures is also very difficult to accomplish (Pastor

et al., 2003).

The compactness and complexity of lignocellulose makes it much more challenging to

enzymatically degrade to fermentable sugars than is the case for starch. Therefore, the cost

of producing a gallon of ethanol from biomass is higher than producing it from starch

(Wyman, 2003).

The effort taken by the U. S. Department of Energy‟s Biomass Program focuses on the

feedstock supply of lignocellulosic biomass such as corn stover, straw, and wood that can be

converted into energy products (i.e. fuels, chemicals, and power) through sugar or

thermochemical platforms. Analysts for the biomass program estimate that 512 million dry

tons of biomass, equivalent to 8.09 quads of primary energy, could initially be available at

less than $50/dry ton delivered (Walsh et al., 2003).

1.3.1 Sugar platform

The conversion of lignocellulosic material to ethanol includes three basic steps:

pretreatment of lignocellulosic materials, hydrolysis of cellulose to fermentable sugars, and

fermentation of sugars to ethanol. Researchers working on the sugar platform of the are

currently focusing on developing technology to remove lignin and break down cellulose and

hemicellulose to their component sugars, including glucose, xylose, mannose, galactose,

9

and chemicals by itself or be burned for electricity. Proposed biorefineries can then

biologically process produced sugars to fuel ethanol or chemical building blocks (EERE,

2006).

The pretreatment step alters the structure of the lignocellulosic material by removing the

lignin and reducing cellulose crystallinity, thus exposing the cellulose for hydrolysis (Mosier

et al., 2005). There are three major approaches for pretreating the biomass: 1) acid

prehydrolysis (low temperature/ concentrated acid and high temperature/dilute acid), 2)

alkaline pretreatment, and a 3) high pressure/high temperature treatment. In the low

temperature, concentrated acid hydrolysis, concentrated acid disrupts the hydrogen bonding

between the cellulose chains. This “decrystallization” forms a homogeneous gelatin with the

acid, and causes the cellulose to be extremely susceptible to hydrolysis (Sheenan and

Himmel, 1999). Research and development of this process has been around since the early

1900‟s, but in the last 20 years it has become increasingly active. At Purdue University and

at Tennessee Valley Authority, researchers have made improvements on the recycling of

concentrated sulfuric acid (Tsao et al., 1982; Broder et al., 1992). However, analysis of

commercialization of these processes proves to be uneconomical due to the high volumes of

acid required (Wright and d'Agincourt, 1984). Even more recently, Arkenol has developed a

patented process in which the decrystallization is followed by an additional hydrolysis with

diluted acid (EERE, 2006). A triple effect evaporator is required to reconcentrate the acid

(Farone and Cuzens, 1996). This process, however, uses excessive amounts of acid which is

10

The dilute acid hydrolysis process in the sugar platform attempts to optimize sugar yields

with two stages of hydrolysis. The first and milder stage of hydrolysis breaks down the

hemicellulose, while the second stage focuses on the more stable cellulose portion of the

lignocellulose (Harris et al., 1985). In 2000, British Columbia International and the DOE‟s

Office of Fuels developed a plan to construct a biomass-to-ethanol plant capable of

producing 20 million gallons of ethanol a year using a dilute acid hydrolysis process (EERE,

2006). Also, Tembec and Georgia Pacific are both operating sulfite pulp mills in North

America, which utilize this dilute acid hydrolysis process to dissolve hemicellulose and

lignin from wood, and produce specialty cellulose pulp (EERE, 2006).

The alkaline pretreatment process involves the addition of a strong base to the

lignocellulosic biomass, causing disruption of the intercellular bonds which crosslink

hemicelluloses, lignin, and cellulose. This results in decrystallinity and separation of the

three components (Sun and Cheng, 2002). The most widely studied base is sodium

hydroxide. Research has shown that at 120°C, lignin content can be reduced by over 55%

within 30 minutes of pretreatment when using 0.5 % NaOH solution (Soto et al., 1994). The

use of lime (calcium hydroxide) as an alkali pretreatment has also been used due its low cost.

Research has shown that compared to an untreated sample, samples treated with lime

increased the rate of enzymatic hydrolysis of corn stover by almost ten times (Kaar and

Holtzapple, 2000). As in acid pretreatment, however, the recovery of reagents is necessary

and adds cost to the overall process.

Once the three main components of lignocellulosic biomass are separated, enzymatic

11

using different enzymes. The three cellulase enzymes used are endoglucanase, exoglucanase,

and β-glucosidase (Sun and Cheng, 2002). To break down the hemicelluloses, xylanases and

β-xylosidases are used. The key economic barrier in this process is the high cost of enzymes

needed to hydrolyze the celluloses. The Biomass Program is currently working with

Genencor International and Novozymes to achieve a 10 to 50 fold reduction in the cost of

these enzymes to enable this platform to be economically competitive with fermentation of

starch or sugar crops (EERE, 2006). Because environmentally friendly enzymes are used,

subsequent fermentation is ideal due to minimal side products produced. However, acid

from the lignin pretreatment process still creates disposal issues.

1.3.2 Thermochemical Platform

The thermochemical platform of the DOE‟s Biomass Program describes biomass

gasification as an approach to producing fuels and chemicals. If biomass is heated with little

or no oxygen, less than needed for combustion, it reduces into a mixture of carbon and

hydrogen in the form of CO

and H

. The resulting gaseous components are called syngas.

This syngas burns more efficiently than biomass and can also be burned in gas turbines

(EERE, 2006). Moreover, if these gasifiers are placed in series, researchers have shown that

they can burn biomass to produce mixtures of gases including the single-carbon gases carbon

dioxide and carbon monoxide, which can be fermented by select microorganisms or mixed

with chemical catalysts for conversion to valuable fuels and chemicals (Huhnke et al., 2002;

Cotter, 2006). The Fischer-Tropsch process uses syngas to create valuable products,

specifically fuels needed for transportation. It uses a transition metal catalyst to convert CO

12

however, are relatively inefficient, and must be further researched to decrease unwanted

byproducts such as methane and methanol (EERE, 2006).

1.4 Starch-based Feedstocks

Starch is the most widespread carbon reserve stored in plants and is of significant

importance in the industry for food, chemical, and enzymatic uses (Geigenberger, 2003). It

is synthesized in plants as a result of photosynthesis, and its composition varies with different

species. While research and development of lignocellulosic biomass conversion processes is

essential to this country‟s movement towards replacing fossil fuels with renewable energy,

more near-term emphasis should be placed on finding alternate and abundant starch sources

that can be implemented within the next three to five years.

1.4.1 Starch Crops

The four main industrial sources of starch today are corn, tapioca, potato, and wheat (van

der Maarel., 2002). As previously stated, corn is the most widely grown source of starch in

the United States for the production of ethanol, but it is concentrated in the Midwestern

states. Steps need to be taken in order to utilize land in other parts of the country. Root and

tuber crops contain on average 70-80% water, 16-24% starch, and trace quantities of proteins

and lipids (Hoover, 2001). Sweetpotatoes (

Ipomoea batatas,

Morning-glory family) are

another major starch-based crop grown in the United States which offers an alternative

material that can be converted to sugars needed for ethanol production and/or value added

products. They are grown primarily in the Southeastern region of the United States and offer

up to a 30% yield in starch, and could be used in addition to corn for renewable sugar

13

alone produced over 80% of the country‟s sweetpotatoes in 2007, growing over 100,600

acres (NASS, 2007). High starch industrial sweetpotatoes (ISPs) have been bred for higher

dry matter content to generate up to 50% higher starch yields than the common

orange-fleshed sweetpotato and are not intended for use as a food crop (Nichols, 2007).

Sweetpotatoes rank as the fifth most important food crop on a fresh-weight basis in

developing countries, after rice, wheat, corn and cassava (Huntrods, 2008). Yet because they

are a low impact crop which is resistant to drought, typhoons, pests and diseases, and can

grow in poor soils, there is greater potential for growing them in various regions and for

purposes outside of food, like the ISP (Noda et al., 1992). Sweetpotatoes have been

considered a good substrate for alcohol fermentation since they have a higher starch yield per

unit land cultivated than grain, and continue to increase in weight during its long growing

season until harvested (Sachs, 1980; Wu and Bagby, 1987).

1.4.2 Starch Composition

Starch is a well-known polymer of glucose, linked by glycosidic bonds. The two major

types of molecules in starch are amylose and amylopectin (van der Maarel et al., 2002).

Amylose, the minor linear polymer in sweetpotatoes, consists mainly of α-1,4 linked

D-glucopyranosol residues (Hoover, 2001). It contains up to 6000 glucose units. Amylopectin,

the major polymer in sweetpotatoes, is composed of short α-1,4 linked linear chains of 10-60

glucose units and α-1,6 linked side chains containing 15-45 glucose units. Amylopectin

contributes to the side branching of starch. One molecule of amylopectin usually contains

14

(van der Maarel et al., 2002). The structure of amylose and amylopectin allow starch to be

converted to glucose, a major sugar feedstock in ethanol conversion.

1.4.3 Starch Conversion

Starch, unlike lignocellulose biomass, is more easily convertible to ethanol and can be

described in three basic steps. Initially, the starch must be converted to a useable

fermentable sugar. This can be achieved through a variety of different processes, including

acid hydrolysis, high temperature and pressure extrusion, and enzymatic hydrolysis. Once

the starch is converted to simple sugars, it must then be fermented using biocatalysts such as

yeast or bacteria to produce ethanol (EERE, 2006). Finally, the aqueous ethanol solution

must be distilled to extract its pure form.

Currently, there are two processes used to create ethanol from corn, the wet milling and

the dry milling processes. Wet milling accounts for about 21% of ethanol production and dry

milling accounts for about 79% of production (USDA, 2006). In the wet milling process,

corn is separated into starch, protein, fiber, and corn germ. Only the pure starch is used for

ethanol production. The rest of the corn is used to make various byproducts, such as corn oil,

corn gluten meal, corn gluten feed. In the dry milling process, corn kernels are ground and

mixed with water. This is then cooked in a process called liquefaction, and enzymes are

added to convert the starch in the corn to glucose in a process called saccharification. The

glucose is then converted to alcohol through fermentation (USDA, 2006). Although there are

fewer useful byproducts produced when using the dry-milling process, it is more efficient.

The need for alternative starch crops will create a demand for the development of

15

is a potential to develop this process using sweetpotatoes as the starch source to produce

fermentable sugars for ethanol and valuable chemicals.

1.5 Acid hydrolysis

Acid hydrolysis of starch uses different concentrations of common acids, primarily HCl,

to hydrolyze starch into fermentable sugars. This is one of the oldest forms of conversion but

has problems with disposal because of the high concentrations of acid used. If all of the acid

is not recovered, it is disposed of with the wastewater runoff. Lee et al. (1985) hydrolyzed

raw cassava starch using a combination of acid hydrolysis (HCl) and commercial enzymes

(pectin depolymerase, α-amylase, glucoamylase) and showed that treating the starch with 0.5

N HCl for 12 hours in addition to saccharifying the starch with α-amylase and glucoamylase

yielded the maximum alcohol production (95% ethanol yield) and converted 65% of the

starch to sugar. Further increases in acid concentration resulted in decreased ethanol yields.

This was attributed to increased production of hydroxymethylfurfural (HMF), a dehydration

product of D-glucose known to inhibit growth and alcohol fermentation by yeast from acid

hydrolysis. Acid hydrolysis of sweetpotatoes at various temperatures (97, 110, and 129.4ºC)

showed that reducing sugars up to a dextrose equivalent of 84% were quickly formed with

elevated concentrations of HCl (1N, 1.5N, 2N HCl) and increased temperature, but this also

significantly increased the HMF concentration and destroyed the reducing sugars (Kim and

Hamdy, 1985; Azhar and Hamdy, 1981 ).

1.6 Temperature and pressure

Temperature treatments and high pressure extrusions have been researched to determine

16

native alpha and beta amylase enzymes found within sweetpotato starches that have been

known to have important effects on the processing of sweetpotatoes (Doebald et al., 1969).

One study using heat treatments during hydrolysis of sweetpotato mashes found that a

temperature of 70°C produced 18.4% maltose and 12.7% sucrose, however only 3.5%

glucose and 2.0% fructose was recovered (expressed as a percentage of dry matter). In

addition, 35.7% of AIS remained after hydrolysis (McArdle and Bouwkamp, 1986).

Depolymerization of starch using high pressure extrusion by Kim and Handy (1987) showed

that high pressure alone, up to 20,000 psi, produced a dextrose equivalent of only 1.15

(measured as a reducing power of dextrose). Also, this study showed no traces of either

glucose or maltose in the solution containing depolymerized starch, indicating that a very

small concentration of starch was hydrolyzed to fermentable sugars. When high pressure

was combined with heat (97ºC) and acid (3N HCl), 17.1% glucose and 26.1% of total sugars

were recorded, however production of HMF was also detected.

1.7 Enzyme hydrolysis

Enzymatic hydrolysis of starch can offer a simpler and more environmentally friendly

method to produce ethanol. Several commercial enzymes can be used to convert starch into

its simple sugars, including α-amylase, β-amylase, and glucoamylase, α-glucosidase, and

pullulanase. The enzyme α-amylase acts on the inner portions of amylose and amylopectin

to form glucose chains of varying length. Conversely, β-amylase breaks the external glucose

units of amylose and amylopectin to produce maltose and shorter glucose chains.

Glucoamylase produces free glucose by cleaving both α-1,4 and α-1,6 glycosidic bonds on

17

amylopectin, and α-glucosidase cleaves short maltose chains to produce glucose (van der

Maarel, 2002).

The enzymatic hydrolysis of starch can be described in two basic steps: liquefaction and

saccharification. Initially, an aqueous slurry of starch is heated at a temperature high enough

to induce gelatinization and liquefied using a liquefying agent (α-amylase, β-amylase) to

produce short chain sugars (dextrins). The liquefied starch is then saccharified at a lower

temperature in the presence of at least one saccharifying enzyme (glucoamylase, pullulanase,

α-glucosidase) to provide an aqueous solution containing 60-80% weight of the original

starch in the form of fermentable sugars (Muller and Miller, 1981).

Liquefaction

Starch + H

O Dextrins + Maltotriose + C

H

O

(Maltose)

Saccharification

Dextrins + Maltotriose + C

H

O

C

H

O

(Glucose)

One issue which arises during the breakdown of this starch is increased viscosity during

gelatinization. Gelatinization is a transition which is caused by heating a starch source in the

presence of excess water, and causes a radial swelling and diffusion of the excess water into

the starch granule (Hoover, 2001; Stevens and Elton, 1981; Donovan, 1979; Evans and

Haismann, 1982; Biliaderis, 1991). The gelatinization temperature for sweetpotatoes

(

Ipomoea batatas

), occurs at a temperature of 84.6°C (Collado et al., 1999). A solution to

this problem, which is necessary for the breakdown of the starch into its simple sugars, is to

add enough water to overcome the increased viscosity and keep the sample in liquid form.

18

starch is expected and encouraged during enzymatic hydrolysis. This process is known to

disrupt the double helices of amylopectin in the amorphous region of the starch granules.

This increases the accessibility of unraveled chains to for diffusion by the enzyme during

continued heating of hydrolysis (Gunaratne and Hoover, 2002).

Studies on enzymatic hydrolysis have been performed testing different enzymes and

starch sources and have revealed promising results. The conversion of cooked sweetpotato

varieties to ethanol using pectinase, α-amylase, and glucoamylase showed that viscosity

reduction with pectinase (0.1ml/100g of ground, wet, Jewel sweetpotato) is important in

ethanol recovery, and produced 15.8 g glucose/100 ml of solution with a theoretical ethanol

yield of 90% using the HiDry variety (Wu and Bagby, 1987). A study of alcohol

fermentation of uncooked sorghum using glucoamylase preparations from

A. niger

and

Rhizopus

sp. resulted in a 91.9% alcohol yield based on the theoretical value of the starch

content and using 30-35% (w/v) (Thammarutwasik et al., 1985). Finally, when hydrolysis of

sweetpotatoes at 100°C for 30 minutes (autoclaved) followed by saccharification at 66ºC for

60 min using a malt containing α-amylase, β-amylase, and α-glucosidase was studied, starch

conversion was 88% . However, viscosity of the samples was very high due to the high

loading rates (0.3-0.5 solid/liquid), and resulting sugars were also low (5.6%). When malt

was then added prior to cooking, corresponding starch conversion increased to 96%

conversion of total starch content and reducing sugars increased to 10.7% (Hosein and

Mellowes, 1989). However, this required the uneconomical addition of 8.9% malt both

19

Many enzyme based studies focus on conversion of starch for use in food (Ridley et al.,

2005; Koehler and Kays, 1991; Vanden et al., 1986)

. It is necessary to further examine key

amylase enzymes at different loading concentrations and incubation times to determine a

more efficient method to convert starch, specifically industrial sweetpotato starch, to useable

sugars that can then be fermented into bioethanol and high value chemicals.

1.8 Ethanol Fermentation

Once starch is converted to simple sugars, it is fermented using biocatalysts such as yeast

or bacteria to produce ethanol (EERE, 2006).

Fermentation

C

H

O

2C

H

OH

(Ethanol)

+ 2CO

The most common strain of yeast used to complete this process is

Saccharomyces

cerevisiae,

due to its high ethanol tolerances

.

Strains of

Saccharomyces

can generate up to

10-15% v/v ethanol (Boulton, 2001, 109). This process is an additional step following

enzymatic hydrolysis in the overall production of ethanol. It typically requires an additional

vessel for hydrolysate removal and yeast addition as well as the added time to allow

complete fermentation to occur.

However, recently an alternative process called simultaneous saccharification and

fermentation (SSF) has been developed which combines the enzymatic hydrolysis of starch

to glucose and the conversion of this glucose to ethanol by fermentation into one operation

(Sree et al., 1999). SSF has several advantages over separate hydrolysis and fermentation.

Capital investments are reduced due to the elimination of an entire step. In addition, SSF

20

immediately fermented after hydrolysis (Pastor et al., 2003). One challenge that SSF creates

is the fact that optimum temperatures for the yeast (35ºC) and enzymes (40-90ºC) are

different. This restricts temperature variation of hydrolysis and fermentation and may limit

effectiveness (activities) of select enzymes.

1.9 Objectives

The effectiveness of different enzyme treatments on glucose and ethanol production from

industrial sweetpotatoes needs to be fully examined. The overall goal of this project is to

effectively generate the information necessary to define a process for conversion of North

Carolina industrial sweetpotato variety FTA-94 (ISPs) into ethanol. This will enable further

improvements to be made in enzyme function and cost, processing, scale up, and breeding.

Specific objectives will be to: 1) Examine the effects of different enzyme loadings on

sugar production during liquefaction with α-amylase; 2) Investigate the effects of solid

loading and different enzyme treatments on sugar production during saccharification with

glucoamylase and pullulanase; 3) Determine ethanol production yields from separate

hydrolysis and fermentation (SHF); and 4) Compare the sugar and ethanol production

between fresh and flour (dried) ISP preparations.

The project is supported by Novozymes North America, Inc. as part of their interest in

defining new uses for enzymes within the fuel ethanol industry. The data collected will

allow research to begin to improve process efficiency/cost and large scale development.

Characterization of a practical process for converting sweetpotatoes to ethanol has potential

21

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